JP2001508054A - HBsAg-derived annexin V binding polypeptide and uses thereof - Google Patents

Abstract

  (57) [Summary] Described are hepatitis B surface antigen-derived polypeptides that can compete with the hepatitis B surface antigen / annexin V interaction or bind to compounds or antibodies that compete with the hepatitis B surface antigen / annexin V interaction. Also disclosed are the use of these polypeptides and antibodies thereto for the diagnosis, treatment and vaccination of infection with hepatitis B virus and / or hepatitis delta virus.

Description

DETAILED DESCRIPTION OF THE INVENTION HBsAg-derived annexin V binding polypeptide and uses thereof Field of the invention     The present invention relates to a hepatitis B virus envelope glycoprotein called hepatitis B surface antigen. For polypeptides derived from proteins, this comprises a hepatitis B surface antigen and a phospholipid binding protein. Compete with the interaction between protein annexin V. The present invention also provides for these Hepatitis B virus and delta hepatitis virus Also for use in diagnosis, treatment and vaccination against I do. Background of the Invention   Hepatitis B virus (HBV) is characterized by marked hepatotropism and species specificity Belong to the family Hepadnaviridae. Delta hepatitis virus (HDV) Represents the HBV subviral satellite that arises (Rizzetto et al., 1986). H BV causes major medical problems such as chronic liver disease and hepatocellular carcinoma (Schroder and Zentgraf, 1990). HDV coinfection is usually Severe compared to HBV infection alone. 300 million people worldwide have been infected with this virus It is estimated that there are owners and only 70,000 holders in the United States are HD Co-infected with V.   Within the HBV genome, more particularly within the S gene (see also the next paragraph) There are natural sequence variants. HBV genotypes AF are based on this sequence diversity. (For a review, see Magnius and Norder, 1 995).   The HBV envelope is a product of the S gene, hepatitis B surface antigen (HBsA g) consists of the following three related glycoproteins, called g): 1) 226 "Small" transmembrane protein (major protein or 2) small S protein and pre-S2 of S gene Corresponds to the area A "medium" protein containing 55 additional aa at the N-terminus, and 3) "Large" protein consisting of 389 or 400 aa corresponding to region: S + PreS2 + preS1 (108-119 N-terminal aa) (Heerman et al., 1 984; Robinson et al., 1987). The HDV envelope is also a whole Derived from HBV, mainly small HbsAg, 5-10% of medium HbsAg and large HbsAg Consists of less than 1% of Ag (Bonino et al., 1986).   The region composed of aa100-160 of "small" HBsAg is outside the virus. It is predicted to be located on the surface (for a review, see Berting et al., 19). 95). Current vaccines using recombinant "small" HBsAg are mainly Critical "a" determinant: for a limited number of epitopes such as aa139-147 Resulting in a protective antibody response. The latter determinant is the region aa100 of "small" HBsAg. -160 has been shown to be the most immunodominant epitope (for review, see See Magnuis and Norder, 1995, and Howard and And Allison, 1995). On the other hand, the majority for the "a" determinant Vaccine escape mutants are now available (Howard and Allison, 1995). Therefore , A new vaccine that can be used in the design of new vaccines against hepatitis B There is a need to characterize epitopes.   Annexin V (endonexin II, placental anticoagulant protein, PP4 or Pocortin V) is a structurally related C known as annexin. a²⁺Is a member of the family of dependent phospholipid binding proteins, (Klee, 1988; Zaks and C reutz, 1990). Annexin V is found in liver, spleen, lung, intestine and placenta (Walker et al., 1990). This protein Quality is Ca²⁺Binds to placental membranes in a dependent manner (Haigler et al., 1987) and Liquid coagulation (Grundman et al., 1988) and phospholippers in vitro. Inhibition of ZeA2 activity (Pepinsky et al., 1988) has been described. Other researchers have reported that Annexin V behaves like an integral membrane protein, Selective Cation Cha (Rojas et al., 1990; Bianch). i et al., 1992).   We have recently found that Annexin V, present on the human liver plasma membrane, has been Ca to sAg²⁺Specific binding in a dependent manner (Hertogs et al. , 1993; WO 94/01554). Interaction between HBsAg and Annexin V The receptor-ligand relationship can be either native human liver annexin V or recombinant annexin V Rabbit immunized with, or rabbit anti-annexin V IgG F (ab ')_TwoH Chicken immunized with fragment is an anti-idio that specifically recognizes HBsAg Further supported by observations that spontaneously develop type antibodies (Ab2) (Hertog et al., 1994). The inventors have also noted that HDV particles can Demonstrated that it binds to Annexin V via HbsAg containing envelope (De Bruin et al., 1994). However, some HbsAg-derived Are described (EP 047710; EP 0155146; WO 9516704), the exact site on HBsAg that binds to Annexin V is Unknown. By mapping this site, both HBV and HDV infection Development of new epitopes that can be used in designing cycle interruption methods Can be led. Purpose of the invention   From the references cited above, a vaccine for the major "a" determinant of "small" HBsAg The emergence of tin escape mutants is due to vaccines currently used for HBV infection. Clearly raise the issue of defense provided by In fact, Wakuchi HBV infection in vaccinated children has been described (Howard et al., 1992). 95). The purpose of the present invention is generally to provide a vaccine or any other composition. Better against hepatitis B (and consequently hepatitis D) infection when used It is to provide a new polypeptide which gives a great protection.   Polypeptides used in vaccine compositions that can provide better protection May be derived from viral antigens that play an important role in virus survival Is high. Annexin V binds to HBsAg, and thus is responsible for HBV and HDV infection. To start Has been demonstrated to play an important role in HBsAg. The syn-V binding domain may not undergo major mutation. This avoids numerous In contrast to the "a" determinant on HBsAg where the variants have already been characterized (Carman et al., 1990). In other words, the annexin V binding domain of HBsAg The development of escape mutants in Maine is unlikely. Therefore, the object of the present invention is Competes with HBsAg / Annexin V interaction or HBsAg / Annexin HB that binds to a compound or antibody that competes with the Syn-V interaction and is immunogenic An object of the present invention is to provide an sAg-derived polypeptide. More specifically, the purpose of the present invention is to , The following sequences: FAKYLWEWASVR, KTCTTPAQGN and TTP The above port containing less than 61 amino acids, including at least 4 amino acids of one of AQGN. To provide a repeptide. In this regard, the identity of such polypeptides It should be clear that ping is not a trivial task. Especially when immunizing Elicits specific responses to annexin V binding epitopes or portions thereof Processing polypeptides in this manner is not at all obvious.   A further object of the present invention is the use of the sequence KTCTTPAQGN or TTPAQGN And the sequence FAKYWEWASVR, or a functionally equivalent of these sequences The purpose is to provide a polypeptide comprising a part or variant.   Another object of the present invention is to provide a vaccine composition comprising the above-mentioned polypeptide as an active ingredient. It is to provide things.   Furthermore, an object of the present invention is to provide HBV and / or HDV or any of them. For vaccinating humans against infection with a mutant strain of HBV and // Vaccine therapeutically for human carriers of HDV or any mutant thereof Providing a vaccine composition as described above for use as an inoculum to inoculate That is. The vaccine composition described above is used to therapeutically vaccinate HBV carriers. The HBV-derived polypeptide can be inoculated with this polypeptide. Elicits a specific antibody response to tides, but in control chimpanzees Natural HBV infection (see Example 5) must result in the production of such antibodies Based on the surprising observations of the present inventors. In other words, HBV infection Vaccine between the above Inoculation of the composition results in a better immune response, and thus better protection.   Accordingly, an object of the present invention is also to provide the above polypeptides upon inoculation in a mammalian host. Peptides, especially the sequence, KTCTTPAQGN or a part thereof, the sequence TTPAQG N or a portion thereof, or the sequence FAKYWEWASVR or a portion thereof The above polypeptide or any of the above, which results in the production of specifically binding antibodies Is to provide a variant of   An object of the present invention is also directed to a polypeptide according to any of the above objects, which is negatively charged. To provide a combination with a modified phospholipid.   Furthermore, an object of the present invention is a combination of any of the above defined polypeptides. It is to provide a polypeptide composition comprising a strain.   It is also an object of the present invention to specifically bind to a polypeptide as defined above, Antibodies or fragments thereof that inhibit the binding of the polypeptide to Annexin V Is to provide.   A further object of the present invention is to provide HBV and / or HDV or any of them. Antibodies as defined above for use in a method of treating humans infected with a variant of Or a pharmaceutical composition comprising a fragment thereof as an active ingredient. .   Further, the object of the present invention is a) The biological sample to be analyzed for the presence of the HBsAg antibody was defined above Contacting with a polypeptide, b) detecting the immunological complex formed between the antibody and the polypeptide H using a polypeptide as defined above, characterized in that Can compete with BsAg / Annexin V interaction and is present in biological samples It is to provide a method for detecting an antibody.   The purpose of the present invention is also to provide HBV and / or HDV or any modification thereof. Polypep as defined above for use in a method of treating a human infected with a heterologous strain An object of the present invention is to provide a pharmaceutical composition containing a tide as an active ingredient.   Further, it is an object of the present invention to provide a binding between Annexin V and the above polypeptide. For use in screening methods for blocking drugs, see above. Definition To provide a modified polypeptide.   The invention also relates to hepatitis B virus and / or hepatitis delta virus or Use as a medicament for treating a human infected with any of the mutants. To provide a polypeptide as defined above.   Finally, the object of the present invention is to provide at least one microplate, as defined above Polypeptide, an appropriate buffer, and the antibody of the polypeptide in a human serum sample. Blocking and washing solutions that favor binding, as well as antibodies in human serum samples A suitable marker that allows the measurement of the complex formed between the body and its polypeptide For in vitro measurement of antibodies to HBsAg present in human serum, including car To provide a kit.   All objects of the invention are believed to be consistent with the embodiments described below. Brief description of drawings and tables   Table 1 shows the "Small" that determines the four genotypes of HBsAg (A, B, C and D). For the region aa99-169 of HBsAg, and Annexin V and Binding to some antibodies and its immunogenicity in chimpanzees and rabbits It provides sequence information on the polypeptides studied.   Table 2 shows the peptides for binding to the anti-HBsAg monoclonal C11F5. The relative affinity of the drug. Briefly, coated on microtiter plates Binding of C11F5 to HBsAg was determined by peptide IGP 1076-10 in solution. Compete with 83. The molar concentration of HBsAg itself producing 50% competition is equal to 1 .   FIG. 1. HBs in culture medium of primary cultures of adult hepatocytes infected with HBV. FIG. 4 shows the effect of anti-idiotype antibody (Ab2) on Ag production. Anti-idiota HB of infected cells at 3, 5, 7, 8 and 10 days after infection in the absence of ip antibody sAg production is represented by “〇”. In the presence of uninfected cells or anti-idiotype antibodies The results of HBsAg production in the culture medium of infected cells were indicated by “+” and “▲”, respectively. Represent. The results shown are the average of the results from five experiments.   FIG. 2 shows anti-idies that specifically recognize the annexin V binding domain of HBsAg. Oh Type Antibody (Ab2) Is In Vitro in Primary Cultures of Human Hepatocytes It shows that HBV infection is prevented. Briefly, this figure shows an anti-idiotype antibody In primary cultures of HBV-infected human hepatocytes in the presence and absence of HBV Autoradiography of Southern Blot Analysis of HBV-DNA and Replication Intermediates Represents a system. The integrated HBV-DNA sequence was identified as HepG2.2.15 (HBV gene). Nom transfected HepG2 cell line) (Sells et al., 1987). (Lane 1). HBV-DNA replication intermediate is an anti-idiotype antibody Detected in cells infected with the HBV inoculum in the absence (lane 4). Les Zones 2 and 3 are for experiments without HBV inoculum and for anti-idiotype anti- FIG. 9 shows the results of HBV-DNA detection in a parallel experiment in the presence of a body.   FIG. 3 shows the anti-idiotype antibody (Ab2) mimicking Annexin V in Table 1. 2 shows binding to the indicated polypeptides and “small” HBsAg. Briefly, polypep Pb or HBsAg was adsorbed to a microtiter plate, and this was Incubated after blocking with serial dilutions (1 / 50-1 / 5000) . Ab2 binding using peroxidase-conjugated anti-rabbit IgG preparation And visualized. Plate using tetramethylbenzidine as color former Color was developed and the plate was finally read at 450 nm.   FIG. 4 shows the binding of Ab2 to several biotinylated forms of the polypeptides shown in Table 1. Indicates a match. Briefly, streptavidin is adsorbed to a microtiter plate. This was incubated after blocking with the biotinylated peptide, followed by A Incubated with serial dilutions of b2 (1/50 to 1/5000). A The binding of b2 was determined using a peroxidase-conjugated anti-rabbit IgG preparation. Visualized. Color plate using tetramethylbenzidine as color former And finally read the plate at 450 nm.   FIG. 5 shows that HBsAg of either genotype A or D was Ab2 and Annexi. It shows that it binds to V. Briefly, serial dilutions of HBsAg were Coated on a tar plate. After blocking, immunize the plate with Annexin V Rabbit-derived Ab2 (Fig. 5A) or peroxidase-labeled annex V itself (FIG. 5B). Ab2 binding was determined using anti-rabbit IgG per Visualized using oxidase conjugate.   FIG. Phosphatidylserine (white) or phosphatidylcholine (black) stages Horseradish peroxidase (HRP) on solid phase HBsAg in the presence of dilution O) Binding of labeled recombinant human AV.   FIG. To HBsAg in the presence of a serial dilution of mAb against human AV Binding of HRPO-labeled AV (U4C8: open triangle, U1E10: closed triangle, E1E8 : White circle, CM1995: black circle).   Figure 8. Solid phase m in the presence of a 75-fold molar excess of unlabeled rat or human AV Positive control of HRPO-labeled AV binding to Ab (without competitor) Relative inhibition (CM1995: open bar, U4C) 8: hatched bar, U1E10: black bar).   FIG. Excess phosphatidylserine (white bar) or phosphatidylcholine (clinical) Binding of HRPO-labeled recombinant human AV to the solid-phase mAb in the presence of Phase expressed as% of competition relative to positive control (experiment without competitors) Sympathetic inhibition (phospholipid / mAb ratio is 30 (w / w)).   FIG. Binding between HBsAg and AV and binding sites for all inhibitory antibodies Visual impression.   FIG. 11 shows chimpanzees vaccinated with some of the polypeptides shown in Table 1. FIG. Briefly, streptavidin or HBsAg The streptavidin-coated wells that have been adsorbed to the Peptide (IGP 1103, 1119 and control peptide 10) 30 and 1038), followed by incubation with the vaccine With serial dilutions (1/20 to 1/2560) of inoculated chimpanzee serum Incubated. Conjugation was performed using peroxidase-conjugated anti-human IgG preparation Was visualized using In this regard, human and chimpanzee IgG antibodies Can detect chimpanzee IgG using an anti-human IgG conjugate It should be noted that they show sufficient cross-reactivity to Plate, Tet Lamethylbenzidine The color was used as a color former and the plate was finally read at 450 nm.   FIG. 12 shows vaccines with polypeptides IGP 1103 and 1119 listed in Table 1. 2 shows the evolution of antibody titers in chimpanzees inoculated. Briefly, streptavige Or HBsAg is adsorbed to a microtiter plate, and streptavidin Uncoated wells with biotinylated peptide (IGP 1103, 1119) Chimpanzee serum incubated after blocking and subsequently vaccinated Were incubated with the serial dilutions (1/20 to 1/2560). Join Were visualized using a peroxidase-conjugated anti-human IgG preparation. Step The color is developed using tetramethylbenzidine as a color former and finally The rate was read at 450 nm. The titer compared to the negative control In addition, it expressed as the serum dilution which gives a positive signal.   FIG. 13 shows the antibody response of chimpanzees challenged with HBV. Briefly Adsorb streptavidin or HBsAg to microtiter plate , Streptavidin-coated wells were treated with biotinylated peptide (IGP 110 3, 1119 and control peptides 1030 and 1038). A single incubation of chimpanzee serum, incubated after packing and subsequently vaccinated Incubated with one dilution (1/20). Binding, peroxidase Visualization was performed using a conjugated anti-human IgG preparation. Plate with tetramethi Color was developed using rubenzidine as a color former and finally the plate was run at 450 nm. I read. No specific reactivity with IGP 1119 or 1103 measured Was. Reactivity with control peptides 1030 and 1038 was 1119 and And 1103 always exceeded.   FIG. 14 shows the cross-reactivity of antibodies elicited by genotype A-derived peptides. You. Briefly, streptavidin is adsorbed to a microtiter plate and This is incubated after blocking with the biotinylated peptide, followed by Serial dilutions of Ngzy serum (1/2^Four~ 1/2¹³). H The binding of the pancreatic antibody was determined using a peroxidase-conjugated anti-human IgG preparation. Visualized using Plates using tetramethylbenzidine as color former And let it develop color, Finally, the plate was read at 450 nm. IGP 1030 has no HbsAg Represents the relevant control peptide.   FIG. 15 shows annex to the HBsAg peptide representing amino acid regions 112-169. 3 shows the binding of Syn-V. Briefly, streptavidin conjugates are HBsAg itself or biotinylated peptide IGP coated on 671, 673 and 80 (representing aa regions 112-169), control Peptide binding to peptide IGP 1038 and anti-idiotype antibody (I GP1119) coupled to horseradish peroxidase ) Was evaluated for binding. The specificity of binding of the peptide to Annexin V was 00 μg / ml), which is known to compete with the binding of Annexin V to HBsAg. Demonstrated by adding the anti-Annexin V monoclonal antibody See also Example 7).   FIG. 16 shows a more precise mapping of the Annexin V binding site on HBsAg. Show. Briefly, a new series of peptides to Annexin V (IGP 1189- 1193, IGPs 1190-1192 are heterobranched peptides, see Table 1. ) Were analyzed as described above for FIG.   FIG. 17 further refines the annexin V binding site on HBsAg by direct competition. Indicates a dense mapping. Briefly, HB coated on microtiter plates Binding of Annexin V to sAg was determined by HBsAg as well as peptide IGP in solution.   1119 and IGP 1193 (both peptides are strept Avidin conjugate).   FIG. 18 shows a more precise mapping of the Ab2 epitope. In short, ma The binding of Ab2 coated on the microtiter plate was determined using peptide P4 in solution. Compete with 67-P471.   FIG. 19 shows the epitope of the monoclonal anti-HBsAg antibody C11F5. Indicates mapping. Briefly, peptides are converted to streptavidin complexes. It was adsorbed on a crotiter plate. Binding of C11F5 to peroxidase Visualization was performed using a conjugated goat anti-mouse antibody.   FIG. 20 shows that Annexin V was added to a peptide containing the HBsAg region 158-169. Indicates binding. Briefly, the coupling was performed as described for FIG. Performed at room temperature instead of 37 ° C. This is to select stronger binding peptides is there.   FIG. 21 shows the relative affinities of the peptides for binding to Annexin V. Briefly, annexin to HBsAg coated on microtiter plates V binding competes with a 200 or 20 fold molar excess of peptide in solution.   FIG. 22 shows chimpanzees and rabbits for epitope regions 115-134. 3 shows the mapping of the antibody response of FIG. Briefly, the peptide is conjugated to streptavidin complex. It was adsorbed on a microtiter plate as a combination. Antibody binding to peroxy Visualized using rabbit anti-human or goat anti-rabbit antibody conjugated to dase .   FIG. 23 shows different peptides containing the epitope regions 158-169 (IGP 1 273 and 1274) are shown. Succinctly Converts peptides into streptavidin complexes in microtiter plates Adsorbed. Antibody binding was determined by rabbit anti-human or peroxidase-conjugated Visualization was performed using a goat anti-rabbit antibody. Detailed description of the invention   The invention described herein incorporates previously published work and pending patent applications. Use. For example, such research may be in scientific literature, patents or pending patent applications. Consists of All these publications and applications cited above or below are hereby incorporated by reference. Incorporated by reference in the handbook.   The present invention provides a method for competing with or interacting with the HBsAg / Annexin V interaction. HBsAg derived, which binds to a compound or antibody that competes with the action and is immunogenic Based on polypeptide findings. Therefore, this peptide and its Antibodies can be used to prevent, diagnose or treat HBV and / or HDV infection. Can be used. In this regard, the use of the terms “HBV and / or HDV” Use can be caused by infection alone with HBV or achieved by superinfection with HDV It should be clear that what can be done. On the other hand, infection with HDV is Not in Germany. In other words, the polypeptides of the present invention and antibodies thereto are HBV-infected alone. Or to prevent, diagnose or treat mixed HBV / HDV infection Can be.   HBsAg is a known antigen (Heerman et al., 1984; Robins on et al., 1987). Based on this finding, the HBsAg-derived Polypeptides can be prepared as described in Houbenweyl (1974) and Atherton. And classical chemical synthesis described by Shepard (1989) By any method known in the art or by Maniatis et al. (198 It can be produced by the recombinant DNA technology described in 2). Honcho As used herein, the term “HBsAg-derived polypeptide” refers to “small” HBsA Polypeptide having an aa sequence equal to or similar to a portion of the aa sequence of g Refers to tide. “HBsAg-derived polypeptide” refers to any genotype of HBV ( Genotypes AF, see Example 6). Should be. In addition, the term "polypeptide" in its sequence is greater than HBsAg Polymers of aa containing less aa, more particularly preferentially 226, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, Polypep containing less than 100, 90 or 80 aa, most preferably less than 70 aa Refers to tide. This term refers to glycosylation, acetylation, phosphorylation, It does not mention or exclude post-translational modifications of the polypeptide, such as decoration. In the definition: For example, a polypeptide comprising one or more analogs of aa, including unnatural aa, Polypeptides having substitution bonds, mutant or natural sequence variants of the polypeptides ( Corresponding to the HBV genotypes AF shown above), Includes polypeptides that contain amide bonds, and other modifications known in the art .   Polypeptides such as those described above include, in particular, annexin as described in the Examples. Binds to an anti-idiotype antibody (Ab2) that competes with HBsAg binding to V Or they bind directly to Annexin V.   The expression "compete with the HBsAg / Annexin V interaction or Binds to compounds or antibodies that compete with the Annexin V interaction " og s et al. (1993 and 1994). This expression is In addition, the polypeptide as defined above interacts with or binds to Annexin V (both Can be used interchangeably). It is. Furthermore, the expression "compounds that compete with the HBsAg / Annexin V interaction Or an antibody "can compete with the HBsAg / Annexin V interaction. Refers to a small molecule. More specifically, the latter term refers to anti-idea as described in the Examples. Refers to a biotype antibody.   The term "immunogenic polypeptide" refers to a polypeptide that provokes an immune response, such as antibody production. Regarding the ability of the repeptide. The term further includes that the polypeptide is a B cell and / or Or T cell epitopes.   More specifically, the present invention preferentially includes 61, 55, 51, 45, 41, 35, Comprising less than 31 or 25 aa, most preferably less than 21 aa, and one of the following sequences: For a polypeptide as defined above, including: KTCTTPAQGN (sequence No. 2, also referred to as “aa 122-131”) or 9, 8, 7, 6, 5 4aa, or FAKYLWEWASVR (SEQ ID NO: 35, “aa158- 169 ”) or 11, 10, 9, 8, 7, 6, 5 or 4aa thereof. Or TTPAQGN (SEQ ID NO: 37, also referred to as “aa125-131”) Is 6, 5 or 4aa. More particularly, the present invention relates to the above-defined sequence a a122-131 (SEQ ID NO: 2) derived polypeptide comprising 4-9aa , For peptides having the following aa sequences: aa 122-125, aa 123 -126, aa124-127, aa125-128, aa126-129, a a127-130, aa128-131, aa122-126, aa123-1 27, aa124-128, aa125-129, aa126-130, aa1 27-131, aa 122-127, aa 123-128, aa 124-129 , Aa 125-130, aa 126-131, aa 122-128, aa 123 -129, aa124-130, aa125-131, aa122-129, a a123-130, aa124-131, aa122-130, aa123-1 31 and aa 122-131. In this regard, the present invention preferably comprises aa12 5-131 (SEQ ID NO: 37), aa125-1 28, aa 125-129 and aa 125-130. Should be. Similarly, the invention relates to the sequence FAKYLWEWASV as defined above. R (also referred to as “aa158-169”) (SEQ ID NO: 35). For a polypeptide having the following aa sequence: 8-161, aa159-162, aa160-163, aa161-164, aa162-165, aa163-166, aa164-167, aa165- 168, aa166-169, aa158-162, aa159-163, aa 160-164, aa161-165, aa162-166, aa163-16 7, aa164-168, aa165-169, aa158-163, aa15 9-164, aa160-165, aa161-166, aa162-167, aa163-168, aa164-169, aa158-164, aa159- 165, aa160-166, aa161-167, aa162-168, aa 163-169, aa158-165, aa159-166, aa160-16 7, aa 161-168, aa 162-169, aa 158-166, aa 15 9-167, aa160-168, aa161-169, aa158-167, aa159-168, aa160-169, 158-168 and aa159- 169. In addition, 1) 4 to 4 derived from the sequence KTCTTPAQGN (SEQ ID NO: 2) Polypeptide comprising 9aa and the sequence FAKYLWEWASVR (SEQ ID NO: 35) ) Comprising 4 to 11 aa derived from) and forming a binding region to Annexin V The peptide can be mapped by any method known in the art, such as the methods described above. 2) Annexin V formed by the latter polypeptide The binding region to may be a conformational binding region, and thus has a continuous a It should also be noted that it cannot be composed of an a-array.   In this regard, the invention relates to the sequence KTCTTPAQGN (SEQ ID NO: 2) or T TPAQGN (SEQ ID NO: 37) and the sequence FAKYWEWESVR (SEQ ID NO: No. 35), or a functionally equivalent part or modification of the sequence thereof. About transformation. The term “functionally equivalent part or variant of the sequence” refers to the hepatitis surface. Compete with antigen / annexin V interaction or hepatitis B surface antigen / annex N V interaction SEQ ID NOs: 2, 35 and 37 bind to compounds or antibodies that compete with Any of the variants or fragments of the peptide represented by Use of the latter The terms, but not exclusive, include glycosylation, acetylation, phosphorylation, fatty acids No specific reference is made to post-translational modifications of the peptide, such as modifications at In the definition A peptide comprising one or more analogs of aa (including non-naturally occurring aa); Peptide having exchange bond, mutant form of peptide or variant of natural sequence (HBV residue) Peptide corresponding to the disulfide bond between cysteine residues) Includes tinylated peptides as well as other modifications known in the art.   The present invention further provides polypeptides, particularly the following, upon inoculation in a mammalian host: Sequence, KTCTTPAQGN (SEQ ID NO: 2) or a part thereof, TTPAQGN (SEQ ID NO: 37) or a portion thereof, or FAKYLWEWASVR (SEQ ID NO: 35) or a portion thereof, resulting in the production of antibodies that specifically bind to one of the above. As defined above.   We have surprisingly found that it is involved in Annexin V binding and is immunogenic. It should be noted that certain polypeptides derived from HBsAg have been found. . First, we succeeded in finding an annexin V binding region on HBsAg. There is no reasonable expectation that, secondly, the peptides defined above may be those of HBsAg Not immunogenic when presented to the immune system as a part, but presented as a peptide Because it is immunogenic only when performed (see Example 3).   The invention also relates to a polypeptide as defined above, such as phosphatidylserine. A combination with a highly negatively charged phospholipid. Phosphatidylserine and A- The interaction with V is demonstrated in the examples. The above polypeptide and the above negative load Combinations with charged phospholipid components can be, for example, covalent or non-covalent Any known in the art, such as in the form of a liposome or a liposome Style may be used.   The invention further relates to polypeptides comprising any combination of the above polypeptides. It relates to a tide composition. The term “polypeptide composition” refers to the same or different arrangements. Any possible mixture of the above polypeptides having a sequence, or the same Is different Any possible linkage (covalently or otherwise) between the above polypeptides having different sequences Other). Examples of the latter polypeptide compositions are simple mixtures, homo or hetero. Telo-branched peptide, streptavidin, avidin or neutravidin ( Neutravidin), a combination of biotinylated peptides, Cross-linked peptide, condensed peptide with or without a linker And recombinantly produced peptides.   The present invention also relates to HBV and / or HDV or any mutant thereof. To vaccinate humans against infection with HBV and / or For therapeutically vaccinating human carriers of HDV or any variant thereof. As an active ingredient that can be used as an inoculum for A vaccine composition comprising the peptide.   The term “vaccine composition” refers to HBV and / or HD partially or completely. Immunogenic compositions capable of inducing protection against V. The term "as an active ingredient" Are components of a vaccine composition that elicits protection against HBV and / or HDV About. The active ingredient (ie, the polypeptide of the present invention) is itself And / or manufacture in an unified form (as described in WO 93/18054) (Molecular Probes Inc., Eugene, OR) Use complexed with Neutralite Avidin according to the instructions be able to. A "vaccine composition" is a composition that, in addition to the active ingredient, Appropriate excipients that do not induce harmful antibody production or protection in the recipient It should also be noted that it contains a diluent, carrier and / or adjuvant. is there. Typically, suitable carriers are proteins, polysaccharides, polylactic acids, polyglycosides. Sizes such as luic acid, polymeric aa, aa copolymer and inactive virus particles It is a slowly metabolized macromolecule. Such carriers are well-known to those skilled in the art. composition Preferred adjuvants for enhancing the efficacy of a product include, but are not limited to, Including: aluminum hydroxide, aluminum described in WO 93/19780 In combination with 3-O-deacylated monophosphoryl lipid A, WO 93 Aluminum phosphate described in US Pat. No. 4,606,918 N-acetyl-muramyl-L-thread Onyl-D-isoglutamine, N-acetyl-normuramyl-L-alanyl-D -Isoglutamine, N-acetylmuramyl-L-alanyl-D-isoglutamyl -L-alanine 2 (1'2 'dipalmitoyl-sn-glycero-3-hydroxy Phosphoryloxy) ethylamine, and 2% squalene / Tween 80 Monophosphoryl lipid A, detoxified endotoxin, trehalose-6,6-diamide in emulsion RIBI (I) containing cholate and cell wall skeleton (MPL + TDM + CWS) mmnoChem Research Inc. , Hamilton, MT) . Any of the three components MPL, TDM or CWS, alone or in duplicate They may be used in combination. In addition, Stimulon (Cambridge)   Bioscience, Worcester, MA) or SAF-1 (Sy adjuvants such as ntex) may be used. In addition, complete Freund Adjuvant (CFA) and incomplete Freund's adjuvant (IFA) were It may be used for application and research purposes. "Vaccine composition" further comprises water, Saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances Excipients and diluents that are essentially non-toxic and non-therapeutic, such as preservatives, etc. including. Typically, the vaccine composition comprises either a liquid solution or a suspension, It is manufactured as an injection. Solution on liquid vehicle before injection (or liquid vehicle before injection) Solid forms) may be prepared. Formulation with adjuvant effect May be emulsified or encapsulated in liposomes to enhance Polypeptide , May be incorporated into the immunostimulatory complex together with the saponin (eg Quil) A (ISCOMS)). The vaccine composition comprises an immunologically effective amount of the polypeptide of the invention. Contains tide and any other of the above ingredients. An "immunologically effective amount" is Administration (in a single dose or as part of a series) to prevent or treat Means valid. This amount depends on the health and physical condition of the individual to be treated. Condition, classification of the individual to be treated (eg, non-human primates, primates, etc.) The ability of the individual's immune system to demonstrate an immune response, the degree of protection desired, the formulation of the vaccine, Evaluation of treating physician, strain of HBV infected, co-infection status with HDV and Varies depending on other relevant factors. This amount is expected to fall within a relatively wide range. This can be determined through conventional trials. Through Usually, this amount is between 0.01 and 1000 μg / dose, more particularly between 0.1 and 100 μg. / Dose with dose. Vaccine compositions may be administered parenterally, typically by injection. It is conventionally administered, for example, subcutaneously or intramuscularly. Further suitable for other modes of administration Formulations include oral formulations and suppositories. Dosing treatment can be up to a single dose schedule. Or a multiple dose schedule. Vaccines should be administered with other immunomodulators. May be given. It should be noted that vaccines may also be useful for treating individuals Where the vaccine is called a "therapeutic vaccine".   The invention further relates to an annexin which specifically binds to a polypeptide as defined above. Antibodies or fragments thereof that inhibit the binding of the polypeptide to V . As used herein, the term “antibody” may be polyclonal or monoclonal. Null antibody. The term "monoclonal antibody" refers to an antibody having a homogeneous antibody population. Refers to a composition. The term is not limiting with respect to the species or source of the antibody. Nor is it intended to be limited by the manner in which it is made. Further terms "Antibodies" can also include at least a portion of an immunoglobulin framework region that is human. Humanized antibodies derived from immunoglobulin sequences, and US Pat. No. 4,946,778 And single-chain antibodies as described in As used herein, the term "(antibody)" Fragment "retains the antigen-binding function and specificity of the parent antibody._ab, F_{(ab) Two} , F_VAnd other fragments. To Annexin V by these antibodies Inhibition of the binding of the above polypeptides is described by Hertogs et al. (1993 and 199). This can be demonstrated in an experiment similar to the one described by 4).   The present invention also relates to HBV and / or HDV or any mutant thereof. It relates to the use of said antibody as a medicament for treating infected humans. the term" "Pharmaceuticals" may include saline, Ringer's solution, dextrose solution , Hanks solution, fixed oil, ethyl oleate, 5% dextrose in saline Substances that enhance isotonicity and chemical stability, such as buffers and preservatives Refers to a composition comprising an antibody according to the present invention in the presence of suitable excipients known to those skilled in the art. . A "pharmaceutical" may be administered by any suitable method within the purview of those skilled in the art. No. The preferred route of administration is parenteral. In parenteral administration, the medicament of the present invention Is defined above Unit, such as a solution, suspension or emulsion, together with pharmaceutically acceptable excipients It is formulated in a dosage injection form. However, dosages and modes of administration depend on the individual. . In general, the medicinal product will be more suitable if the antibody is between 1 μg / kg and 10 mg / kg. Or between 10 μg / kg and 5 mg / kg, most preferably 0.1 mg / kg And 2 mg / kg. Preferably, a medicament The article is given as a bolus dose. Continuous infusion may be used. In that case, the doctor The drug is between 5 and 20 μg / kg / min, more preferably 7 μg / kg / min. / Kg / min and 15 μg / kg / min.   The invention further provides 1) The biological sample to be analyzed for the presence of the HBsAg antibody was defined above Contacting with a polypeptide, 2) detecting an immunological complex formed between the antibody and the polypeptide Can compete with the HBsAg / Annexin V interaction, comprising: And for use in a method of detecting antibodies present in a biological sample, as described above. It relates to the use of the defined polypeptide. As used herein, the term "detection method" Method is an assay that utilizes biotin and avidin or streptavidin Known in the art, such as ELISA, and immunoprecipitation and agglutination assays Refers to any immunoassay. A detailed description of these assays can be found in WO 9 6/13590. In this regard, the present invention also provides at least One microplate, the polypeptide as defined above, the appropriate buffer, Blocking and favorable binding of the polypeptide to antibodies in a human serum sample; A washing solution, formed between the antibody and the polypeptide in a human serum sample; Of antibodies against HBsAg, including appropriate markers that allow the measurement of The present invention relates to a diagnostic kit for in vitro measurement.   The present invention also relates to HBV and / or HDV or any mutant thereof. Use of a polypeptide as defined above as a medicament for treating an infected human About. The term "pharmaceutical" may optionally include suitable excipients known to those of skill in the art. Refers to a composition comprising a polypeptide according to the invention in the presence of Appropriate drug delivery Formulation, throw The administration route and dosage are the same as described above for the pharmaceutical containing the antibody.   The invention finally blocks the binding between Annexin V and said polypeptide. The above defined poly for use in screening methods for drugs It relates to the use of peptides. As used herein, the term "drugs for drugs" The "leaning method" can be any known in the art suitable for drug screening. Refers to an assay. In particular, this term refers to any of the terms described in WO 96/13590. Of the immunoassay. The term “drug” targets a polypeptide as defined above It can refer to any compound that becomes or binds to it.   The invention will now be described by way of examples which describe particularly advantageous embodiments. Only However, these embodiments are illustrative and do not limit the present invention. Example Example 1: Transfection of rat FTO2B cells with human annexin V   To demonstrate the involvement of human AV (hA-V) in the first stages of HBV infection First, two in vitro cell models, primary human hepatocytes and rat hepatocytes Cells were used. Expression of human AV in these cells Using monoclonal antibodies (monoclonal antibody U4C8) Evaluated by estane blotting and immunocytochemistry. Of these cells HBV infectivity is determined by two replication markers: precore / core mRNA detection and And on-close (ccc) DNA detection.   First, the specificity of detecting HBV precore / core mRNA as a replication marker was examined. Debated. Precore / core mRNA was detected in HBV infected liver tissue But not detected in control liver tissue negative for HBV infection Was. The precore / core mRNA is also HBV-DNA transfected HepG. 2.2. In HBV-producing cells from the cell line (Sells et al., 1987) Was detected, but not in PLC / PRF / 5 cells. these Cells contain the integrated HBV genome and produce HBsAg, but No sexual particles are produced. The HBV inoculum used in further experiments was precore / Includes core mRNA First, it contained only HBV-DNA.   The fact that RNA was indeed the template for amplification gives rise to the lack of amplification Confirmed by omitting the RT step or digesting RNAse before cDNA synthesis. H Southern blot hi using BV-DNA specific oligonucleotide probes Hybridization, HBV-infected liver tissue and HBV-DNA PCR product of Sfect HepG 2.2.15 cells (Sells et al., 1987) Contains specific HBV sequences. In control liver tissue, Was not observed. Precore / core mRNA is the result of HBV replication Further confirms that it is not due to contamination of RNA present in HBV particles To detect another replication marker, cccDNA, by PCR. Was. cccDNA was transfected with HBV-infected liver tissue and HBV-DNA Shown in HepG2 cells, but not in inoculum.   The presence of cccDNA is related to the presence of precore / core mRNA.   To demonstrate the involvement of hA-V in the first stages of HBV infection, we First examined the infectivity of primary human hepatocytes to rat hepatocytes.   By immunostaining and Western blotting, human hepatocytes were given hA-V It was shown that rat hepatocytes do not express hA-V. Primary human liver After in vitro infection of the cells, pre-core / co-preparation was performed on days 1-15 post-infection, as examined. MRNA was detected, and HBV-DNA was detected on days 3 to 15 after infection as far as examined. Became detectable in the culture medium. After in vitro infection of rat hepatocytes , Neither precore / core mRNA nor HBV-DNA was detected.   To further determine the involvement of human annexin V in HBV infection, HBV A rat liver cancer FTO2B cell line that is not infected by Transduced with a construct containing the hA-V gene. One of the transduced cell lines was FTO9 . Let it be 1. HA-V tracing was performed by immunostaining and Western blotting. The transfected rat hepatoma cell line FTO9.1 expresses hA-V but retains the original Untransfected rat hepatoma cell line FTO2B does not express hA-V It has been shown. Infectivity of FTO9.1 cell line was detected as early as 1 day after infection HBV pre-core Non-transduced FTO2B cells, as demonstrated by / core mRNA, were examined As far as 7 days post infection, it was negative for HBV precore / core mRNA. Sa In addition, cccDNA was FTO 9.1 from day 1 to day 3 post-infection, as examined. Detected in cells. In addition, HBV-DNA is pre- and post-infection. Was not detected in the culture medium for 2 days, but from the third day to the seventh day It has been detected even to the eyes. HBV-DNA is non-plasmid for up to one week after infection. It was not detected in the culture medium of the transduced FTO2B cells.   We have shown that human annexin V is involved in the first stages of HBV infection, Human annexin V is cell susceptible to HBV infection and species barrier to HBV infection It can be concluded that plays an important role in. Example 2: Anti-idiotype against HBV infection in primary cultures of human hepatocytes Effect of antibody (Ab2)   Human hepatocytes were obtained from human liver tissue obtained from post-mortem donors using previously described (Ri jntjes et al., 1986) isolated by two-step collagen perfusion. Culture Hepatocytes were transfected with infectious human serum (HBV-DNA positive, HBsAg positive, HBeA g positive) by overnight incubation at 37 ° C by incubation with a 1/20 dilution. Followed by extensive washing. Sandwiches using specific monoclonal antibodies ELISA was used for both capture and detection of HBsAg.   As shown in FIG. 1, HBsAg production was determined for humans infected with HBV containing inoculum. It was observed in the culture medium of hepatocytes (n = 5). In contrast, in the presence of Ab2, No such HBsAg production was observed. FIG. 1 further shows the production of HBsAg. Birth was maximal 8 days after infection. Thereafter, HBsAg production was Deterioration of primary cultures of human hepatocytes, as seen in control experiments Attenuated due to the condition.   Presence of HBV replication intermediates in infected cells is an essential marker of successful infection Therefore, total DNA was isolated from primary cultures of human hepatocytes used in these experiments. This was subjected to Southern blot analysis (Maniatis et al., 1982). probe As a control for the specificity of DNA isolated from the HepG 2.2.15 cell line was also examined. As shown in Figure 2 Thus, HBV-DNA and replication intermediates infect the inoculum in the absence of Ab2. Detected in the exposed cells. In contrast, the HBV-DNA sequence uses Ab2 Was not observed in the experiments performed. Example 3 Mapping of Annexin V Binding Site on HBsAg   Consists of aa112-169 to find binding sites on HBsAg The area was considered. This is firstly predicted to be located on the outer surface of the virus Second, anti-idiotype antibody Ab2 (immunized with human annexin V). Of HBsAg with Hertogs et al., 1994) produced in isolated rabbits In the case of HBsAg digestion with endoproteinase-LysC, Because it is Lysine is located in this region of the HBsAg molecule (genotype A) Only found in aa122, aa144 and aa160. This area Three peptides covering were synthesized. The first two peptides are HBsAg molecules Represents the outside of. These peptides are derived from the genotype C sequence. 3rd poly Peptide represents the region outside of HBsAg that has undergone the most nonconservative mutations. This peptide is derived from genotype A.   Since Ab2 binds to polypeptide 3, we believe that this polypeptide It was concluded that it contained a binding epitope on HBsAg for human annexin V.   Based on these results, and HB covering amino acids 139-147 The monoclonal antibody recognizing the sAg major "a" determinant is HBsAg / Annex V The "a" determinant is directly involved in Annexin V binding, as it does not interfere with the interaction. Maybe not. Therefore, we propose that the HBsAg ectodomain (ec Attention was paid to the amino-terminal part of the todomain. "Small" HBsAg aa99 Polypeptides covering the region between and 136 (IGP 1076-1083) , See Table 1). Polypeptide 1077 derived from genotype C And 1080 are mutated (a) to reduce the number of cysteines in the peptide. a107, C to S). Furthermore, they appear to be mutated in a non-conservative manner. (Eg, aa126, T to I and aa131, N to T), HBsA g of four naturally occurring genotypes (A, B, C and D, see Table 1) There are two aa in the region aa99-136 to determine. For this reason, books We have synthesized two other peptides: one is the sequence aa1 from genotype A 15-134 (IGP 1094), the other in C-only position 126 Includes the genotype C mutation (IGP 1093). In this regard, some other natural It should be noted that mutations are also present, but they are usually of a conservative nature. (Aa122, R to K, aa134, F to Y and aa113 and 1 14, S to T).   All of these synthesized polypeptides (IGP 1076-1094) were The reactivity with the idiotypic antibody Ab2 was tested (see figures and tables above). See brief description). Ab2 mimics Annexin V and has IGP 1094 Only bound (FIG. 3). Polypeptide 1094 was also produced a second time and the latter The fruit was confirmed a second time.   The lack of reactivity of other polypeptides covering regions 115-113 is due to 126 And was found to be the result of substitution at positions 131 and 131. In fact, these two 4 based on IGP 1094 representing all combinations of mutations at one position Were synthesized (IGP 1102-1105). The latter polypepti Was synthesized in biotinylated form and purified by RP-HPLC. Then Ab Their reactivity with 2 was measured (see FIG. 4). So we have Show that the binding of the peptide to Ab2 is indeed affected by the latter mutation. Proof: mutation at position 126 (T to I, peptides 1103 and 1102) Has a slight binding Loss, while the mutation at position 131 (N to T, peptides 1104 and 1 105) resulted in almost complete loss of binding to Ab2.   Therefore, the C-terminal region of this polypeptide (aa 125-134) is very heavy. It seems to be important. Because mutations in this region alter the binding to Ab2 Because it can be destroyed. However, within a complete HBsAg molecule, These mutations do not disrupt binding to Annexin V. Because genotype A or D Binding of Annexin V and Ab2 to any of the HBsAg (See FIG. 5). This is probably because the complete molecule is at positions 126 and 131 Adopts a stronger conformation that is not affected by mutations in position Because. On the other hand, within the short polypeptide (20aa), a single mutation More intense conformational changes can occur that affect binding to syn. Example 3 ': Mapping of annexin V binding site on HBsAg (further experiments )   Further determine which portions of polypeptides 115-134 are responsible for binding HBsAg. Then, a 10 amino acid peptide covering this sequence was Synthesized.   These peptides were tested for reactivity with the anti-idiotypic antibody Ab . We propose a peptide covering the region of aa122-131 of HBsAg P468 was found to be able to bind to anti-idiotype antibody (Ab2). This This means that the binding epitope of HBsAg is in the region from aa122 to aa131. Means that it must be located in the area. Ab2 binds HBsAg to AV These experiments have been shown to compete for HBsAg because The nexin V binding domain is between aa122 and aa131, or Indicates that it is located in a conformational vicinity.   Thus, those skilled in the art will appreciate that variants of this region that specifically bind to human annexin V And / or derivatives may be synthesized. Such variants are the pepti discussed above. May include any type of variant. Example 4: Effect of phosphatidylserine on HBsAg-Annexin V binding   Due to the nature of Annexin V, which binds to phospholipids (phosphatidylserine), Consider the possibility that proteins can bind to HBsAg via their lipid components And Because HBsAg lipids account for up to 30% of the volume of HBsAg particles. Because it can occupy. Thus, phosphorylation of Annexin V binding to HBsAg The effects of lipids were studied. Phosphatidylse for binding of AV to HBsAg A clear inhibitory effect of phosphorus was observed, but phosphatidylcholine did not inhibit binding. (See FIG. 6). In addition, transfected rat liver cancer cells In other words, HBV infection of FTO9.1 cells is inhibited by phosphatidylserine. Can harm.   To further elucidate the binding mechanism between HBsAg and Annexin V, the present invention They used a monoclonal antibody against human annexin V. All mA b is of the IgG class. Two mAbs, U4C8 and CM1 995 (Dr. Erik Depla, Innogenetics NV, I ndus tripark 7 box 4, B-9052 Ghent, Belgium ; Phone: +32 9 241 07 11; Fax: +32 9 241 0799) is the binding of Annexin V to HBsAg (See FIG. 7). Markers for monoclonal antibodies Annexin V binding is reduced by unlabeled human liver annexin V. It is not reduced by rat liver annexin V (see FIG. 8). this thing Means that these antibodies are species-specific. The phase between these two antibodies That they do not compete with each other for binding to Annexin V , Which means that they recognize different epitopes. No. Antibody UIE10 (Dr. Erik Depla, Innogenetics) N. V. , Industriepark 7 box 4, B-9052 Gh ent, Belgium; Telephone: +32 9 241 07 11; Fax : +32 9 241 0799) is species-specific And could not compete with HBsAg for binding to Annexin V, In addition, this antibody did not compete with U4C8 and CM1995 and therefore the third antibody on AV Recognize the epitope of   The effect of phospholipids on the binding of these mAbs to Annexin V was then examined. (See FIG. 9). Phosphatidylserine is CM1 to Annexin V Can inhibit the binding of U4C8 to Annexin V. From the finding that they can only be inhibited, we believe that CM1995 Inhibits protein-phospholipid interactions while U4C8 interacts with human annexin V To conclude that it inhibits protein-protein interactions with HBsAg Can be. Furthermore, the interaction of HBsAg with Ab2 is dependent on the type of phospholipid It is not affected at all by quality.   We believe that HBsAg-Annexin V binding is at least a double interaction. Can be concluded. This is the phospholipid binding site of Annexin V Negatively charged phosphorus, such as phosphatidylserine, of HBsAg particles via Binding of Annexin V to lipids and a second different epitope of Annexin V Based on protein-protein binding between HBsAg particles and protein components Second mutual Including action. FIG. 10 gives an overview of this interaction. Example 5: Immunogenicity of sequences aa115-134   Annexin V binding domain of HBsAg was purified by RP-HPLC (96.6% purity) N-terminal biotinylated polypeptide (IGP 1103) and C Tetrameric polypeptides on terminally biotinylated lysine matrix (IGP 11 19, see Table 1). Lysine matrix and desired To obtain a small linker between the sequence and the sequence of peptide 1119, the C-terminal Was further extended using another 2aa of Excludes chemicals resulting from peptide synthesis The latter peptide was also purified by RP-HPLC for removal. Both Avidin complex (Molecular)   Probes, Inc. , Eugene, OR).   -Peptide 1103: 200 μg of peptide was converted to 1.1 mg of Neutral It was mixed with item Avidin in a final volume of 2.5 ml saline. this The solution was stored at -20 <0> C in 500 [mu] l aliquots for vaccine purposes.   Peptide 1119: 832 μg of peptide was added to 1.0 mg of Neutral It was mixed with item Avidin in a final volume of 2.5 ml saline. this The solution was also stored as a 500 μl aliquot at −20 ° C. for vaccine purposes.   The above free peptide and two Neutralite Avidin complexes Were evaluated for binding to Ab2. All results were positive (sun That is, all peptides bound to Ab2). The following protocol for chimpanzees Immunized according to the following: prior to injection into one of the upper arms of the chimpanzee, Neutra Lite Avidin Complexed Peptide as an Adjuvant RIBI (ImmunoChem Research Inc., Ham) ilton, MT); injections were performed on days 0 and 21; blood collection Was performed on days -3, 4, 11, 18, 25 and 32 after immunization.   Both Neutralite Avidin complexed peptides, two controls Roll peptide (ie, similar to IGP 1103 and 1119, respectively) Features [possible IGPs 1038 and 1030) having solubility, biotinylation, branching) and H The antibody response to BsAg was monitored. Distinct and specific reactivity is HBsA g and peptides IGP 1103 and 1119 (FIG. 11). ). In addition, both HBsAg and the peptides IGP 1103 and 1119 Ab titers were increased after the second immunization (FIG. 12). Therefore, these conclusions The result is that the peptide sequence aa115-134 is a B-cell protein that is also present on HBsAg. Demonstrate possession of pitope. Because immunity with aa115-134 This is because the induced Ab specifically recognizes HBsAg.   Challenge the control chimpanzee with the standard inoculum for HBV Was. Serum samples were taken before and after inoculation and were used for HBsAg and peptide IGP. Screening for the Presence of Antibodies that Bind 1103 and IGP 1119 did. The specificity of binding was determined using the same control peptide (IGP 1030 and 1038). Anti-HBsAg cello conversion Are clearly shown, but none of the peptides (IGP 1103, 1119, 1 030 and 1038) were not shown (FIG. 13). . These results indicate that when presented to the immune system as part of HBsAg, HBsAg g B-cell epitope in region aa115-134 not particularly immunogenic Indicates that it may not. Example 6: Cross-reactivity of antibodies elicited by genotype A-derived peptides   Chimpanzee sera immunized with peptides 1103 and 1119 were converted to other genetic Anti-cross-reacting with the same region (aa115-134) of HBsAg from the progeny Body presence was assessed. As judged from FIG. 14, the genotype A-derived pep The antibody response elicited by the tides 1119 and 1103 is genotype B (IG P 1104), genotype C (IGP 1105) and genotype D (IGP It clearly cross-reacts with peptides having non-conservative mutations from 1104). That As a result, it is derived from the same region (aa115-134) of HBsAg but has a different genotype. The peptides that are also derived from the genotype A derived peptides IGP 1103 and 1 Eliciting a cross-reactive immune response similar to that elicited by I.119 Can argue. Example 7: Annex to HBsAg peptide representing amino acid region 112-169 Direct connection of V   HBsAg itself or microtiter as streptavidin complex V to a biotinylated peptide coated on a rate (horseradish peroxy) (Bound to the sidase) was evaluated for binding. HBsAg region 112-169 Represented peptides (IGP 671, 672 and 80), anti-idiotypic antibodies and Binding peptide (IGP 1119) and control peptide (IGP   1038). The binding specificity was determined for excess (100 μg / ml) HBsAg Anti-annexin V monoclonal known to compete with nexin V binding Demonstrated by using an antibody (U4C8, see Example 4). Complete All binding assays were performed using 1 mM CaCl_TwoAnd 1 mM MgCl_TwoTr with added is buffered saline (10 mM tris) 150 mM NaCl, pH 7.5 ) (= TBS_{Ca / mg}). Plate HbsAg or peptide / s After coating with the treptavidin conjugate, nonspecific binding was performed by_{Ca / mg} Low by blocking at 37 ° C with -3% gelatin (fish gelatin) Reduce. Incubation with Annexin V and final competitor was performed with TB S_{Ca / mg}Performed at 37 ° C. in -3% gelatin. After incubation, play To TBS_{Ca / mg}-Washed with 0.05% Tween 20.   The data shown in FIG. 15 shows that peptides IGP 671 and IGP 1119 And binding to nexin V. The latter data is provided by IGP 1119 Confirming the importance of the amino acid regions 115-134 covered, the region aa15 The importance of 8-168 is also demonstrated. This is because the latter Ia covering the aa region GP671 binds to Annexin V. Example 8: Finer mapping of annexin V binding site on HBsAg   Based on the results from Example 7, a new set of peptides was synthesized (IGP 1189-1193). These sequences are concluded from Examples 3, 3 'and 7. A Covers potential epitopes for binding to Nexin V. In addition, potential The pitopes were combined in one molecule as heterobranched peptides (IGP 11 90-1192). The experimental settings were the same as in Example 7, and again Specificity demonstrated by competing for binding with the same monoclonal antibody (See FIG. 16).   The highest binding was achieved with the peptides IGP 1193, 1190, 671 and 1119. Was observed. Therefore, the sequence of IGP 1119 (aa 115-134 ), More particularly the amino-terminal portion of this sequence represented by IGP 1193 (Aa115-125) is a key in the binding of Annexin V to HBsAg Seems to play a role. However, the C-terminal portion of IGP 1119 is specifically In combination with acid region 158-169, binding of Annexin V to HBsAg Seems to be important. The latter is linked to Annexin V in this example. Binding of telo-branched peptide IGP 1190 and amino acid region shown in Example 3 ' Demonstrated by Ab2 binding to peptides covering 122-131. Example 9: Further refinement of annexin V binding site on HBsAg by direct competition mapping   A series of peptides used in Example 8 were used to bind HBsAg to annexin. Analysis of their ability to compete with the Peptide IGP 1119 and 1193 Only were found to compete in the concentration range studied. It in solution Present peptides as streptavidin conjugates to improve their presentation. Was. In this experiment, HBsAg coated on a microtiter plate was The binding of nexin V was determined by HBsAg or peptide IGP 1119 and in solution. And IGP 1193 (both peptides were streptavidin conjugates) As presented). Results (see FIG. 17, molar excess = [competitor in solution] / [Coated HBsAg]), the amino acid region 115-134, especially IGP   The importance of the N-terminal part of this region covered by 1193 was confirmed. Example 10: More precise mapping of Ab2 epitope   The peptides mentioned in Example 3 '(P461-P471) bind to Ab2 For their capabilities, different approaches than the approach used in Example 3 'were used. Further analysis was performed using a roach. In the latter embodiment, the peptide is It was adsorbed on a titer plate and the binding to Ab2 was evaluated. These peptides Do not adsorb equally well to the microtiter plate, This can result in a false negative rating. In this setting, HBsAg itself is Plate, adsorb the peptide, use Ab2 in the solution, Binding to BsAg and finally the binding of Ab2 to HBsAg itself is assessed. this In a manner, partial competition was observed for peptides P468, 489, 470 and 471. (FIG. 18, only results for peptides P467-471 are shown) . This result indicates that the binding site (or at least one of the binding sites) of Ab2 In the region 125-131 (TTPAQGN) which is a common part of these peptides, Enable mapping. This result indicates that this Ab2 epitope Confirm the importance of amino acids 126 and 131. These two amino acids are Genotype-specific reactivity of Ab2 is determined. However, the binding of Ab2 is partially Only genotype specific. Because good reactivity is not observed for genotype D (Example 3, FIG. 5). "Parts" with peptides P468-471 Target (up to 50%, FIG. 18), along with the finding of competition, Tope is involved in Ab2 (and consequently Annexin V) binding to HBsAg The conclusion is that we must do this. This is because AV binding is genotype specific and (Example 3, FIG. 5). Based on the above experiment And identified two candidate regions:   Region 1 aa 115-126   Region 2 aa 158-169 Example 11: Further establishing the role of regions 115-126   In light of the above results, annex to IGP 1193 (aa115-126) Shi Confirmation is required for the coupling of V. Because this peptide is an Ab2 epitope And only two amino acids overlap, thus providing a complete Ab2 / Annexin V binding Because it may be a second epitope that needs to be covered in order to do so. In addition, Region have no amino acids that change in a non-conservative manner according to genotype.   To confirm the results obtained above using this peptide (Examples 8 and 9) A purified peptide was required. The crude peptide was first evaluated by mass spectrometry (MS) Was. The spectrum revealed a whole series of molecules, all of which were expected More than 90% of the peptides, not the size, have a substantially higher molecular weight than expected (Data not shown). For these reasons, the peptide was synthesized again and immediately Was analyzed by MS. A new peptide preparation, designated IGP 1193B, It contained the expected molecule, but did not bind to Annexin V. Region 115 The lack of binding of Annexin V to -126 indicates that this region is represented in different presentation modes. Confirmed by a whole new peptide in the series (branched peptide: IGP 1363, C-terminal biotinylation: IGP 1368).   This result is confirmed by another experimental approach. For HBsAg, Monoclonal antibody C11F that cannot inhibit the binding of Annexin V to HBsAg 5 was mapped (Example 12). The epitope portion of this antibody is located in region 11 9-124, which indicates that this region corresponds to Ab2 and thus Annexin V Further evidence that it does not participate in binding. Example 12: Mapping of other epitopes (monoclonal antibody)   Using peptides from our complete series of HBsAg ectodomains Perform mapping of other determinants such as monoclonal antibody epitopes did. Among the monoclonal antibodies studied, C11F5 (Dr. Erik Depla, Innogenetics N.A. V. , Industriepark 7b ox 4, B-9052 Ghent, Belgium; Telephone: +32 92 Fax: +32 9 241 07 99 Only one could be mapped in detail. All used The reactivity of this monoclonal antibody to the peptide is shown in FIG. Relative of purified peptide 1076-1083 for binding of C11F5 to The competition (for BsAg) is shown in Table 2. Both peptides 1076 and 1081 Shows higher relative competition compared to other peptides. This observation was made in Region 1 Supports the presence of the first part of the epitope at 05-111. 107th place Mutations in the stain result in a relative loss comparable to the loss of this part of the epitope. Results in loss of affinity. The second part of the epitope is located in regions 119-124. (Reactivity with peptides 1083, 1193B and 1275). This combination of epitopes is surprising. Because this was before And in particular this has been described by Chen et al., 1996. Because it does not fit the current structural model of HBsAg, which has been predicted. It Thus, by mapping this epitope, a new structure in the structure of HBsAg Insights are guided. Example 13: Further study of the role of regions 158-169   The role of this part of HBsAg has been described by a series of new peptides (1273, 1274). , 1362, 1367). Access to these peptides The binding of nexin V is shown in FIG. For this experiment, the binding was 158-16 All peptides containing 9 regions and some also derived from HBsAg An additional control peptide was checked. Peptides showing high binding The assay was run at room temperature (3 as in the experiment above) to select for (Not 7 ° C). Interpretation of these results indicates: 1 / The binding to peptides 671 and 1190 observed above was confirmed; 2 / regions 158-167 contain sufficient information to allow annexin V binding To Including: binding to peptides 1367 and 1362; Since only the 3 / C-terminal biotinylated peptide allows binding, the N-terminal Edges need to be available for bonding. This observation confirms the proper presentation of the epitope. Emphasize the importance of presentation. N-terminal biotinylated peptide 1274 binds Annexin V Does not allow for This finding provides a way to present peptides to enable reactivity. Emphasize the importance of This means that 158-1 by the peptide IGP 1362 Confirmation in competition experiments (Figure 21) revealing excellent presentation of 69 epitopes Was. From these experiments, presentation is also important in obtaining good immunoreactivity. It can be concluded that there is. Example 14: Immunogenicity   Peptides 1119, 1273 and 1274 were used for immunization of rabbits. Was. For chimpanzee immunization (Example 5), peptides were injected with Neutrali. Complexed with te avidin. For each peptide, three times in equimolar amounts 100 μg of peptide complexed with Neutralite avidin Injected. A total of six rabbits were used (two rabbits for each peptide) .   Antibody responses were evaluated for cross-reactivity with HBsAg and all Were mapped using the available peptides. For peptide 1119 Elicited immune response by a mixture of IGP 1103 and 1119. A comparison was made against the immune response elicited in the monkey (Example 5). 127 3 (115-126 and 158-169 as branched peptides) and 12 74 (158-169 as an N-terminal biotinylated peptide) The immune responses elicited were also compared. Because both peptides have sequence 15 8-169 in a completely different presentation. From Figure 22, the chimpanzee And immunization of rabbits produce an effective immune response against the complete antigen sequence (I GP 673, 1102, 1103, 1104, 1105, 1119 and HB (sAg itself), it is clear that the recognition of a single epitope is completely different. A No antibodies to the b2 epitope were detected in chimpanzees (eg, , IGP 1189 and Lack of reactivity with 80). However, rabbits immunized with IGP 1119 alone did not Both showed very effective immune responses to this specific epitope. This fruit The main conclusion from the experiment is that IGP 1119 has been shown to produce a desired immune response. It could be much more efficient. Co-immunization with IGP 1103 This resulted in an altered immune response recognizing other epitopes. From this example, the desired Limited to a response epitope (122-131 or 125-131) It is anticipated that even a code will be much more effective at generating the desired response. Wear. Because such peptides were clearly selected in chimpanzee experiments Because it does not allow another reactivity chosen. In addition, IGP 1273 and Presentation of such a minimal epitope, as can be determined from the immunizations at is important.   Comparing rabbit immune responses to IGPs 1273 and 1274, It is immediately apparent that GP 1274 does not show an effective immune response (FIG. 23). ). This is due to the lack of reactivity to HBsAg and very weak Can be judged from the reactivity. However, immunization with IGP 1273 Always produces an effective immune response. Reactivity to HBsAg was observed, indicating that All peptides with 8-169 are recognized, but again this peptide has Some sequences 115-126 did not generate antibodies (eg, IGP 11 Lack of reactivity with 93B, 1363 and 1368). This example shows the structure and It is again shown that the method of presentation and presentation is important for exhibiting the desired immune response. This From these examples, one of ordinary skill in the art will appreciate, for example, and without limitation, branched (e) Mo or hetero (heterobranching can be derived from HBsAg or not) Can be performed by any of the sequences)), biotinylated (with or without spacers) N-terminus without, C-terminus with or without spacer) complexed with avidin-like molecule Attached to unrelated molecules (eg, ovalbumin, hemocyanin) Circularized (eg, C-terminal and N-terminal) The addition of cysteine at the ends) such molecules can be engineered using peptides. It can be stated that.   In summary, the above experiments demonstrate that amino acid regions 122-131 and 158-16 9 or a portion thereof is directly involved in the binding of HBsAg to Annexin V Ming Shown clearly. Therefore, including these regions, or variants or parts thereof, Any new molecules that specifically bind to human annexin V are part of the present invention. You. table 1Table 1 continuedTable 1 continuedTable 2

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) G01N 33/15 G01N 33/15 Z 33/53 33/53 D 33/566 33/566 33/576 33 / 576 B (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ) , CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA ( AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE , DK, EE , ES, FI, GB, GE, GH, HU, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU , ZW (72) Inventor Yap, Singh-Heen Belgium, Bee-3221 Nylaude, Craicant 22th (72) Inventor De Maier, Sandra Belgium, Bey-2340 Beerse, Larens Trat 14

Claims (1)

[Claims] 1. Compete with Hepatitis B surface antigen / Annexin V interaction or Hepatitis B table Binds to a compound or antibody that competes with the surface antigen / annexin V interaction, type B An immunogenic polypeptide derived from hepatitis surface antigen. 2. The following array:   -FAKYWEWESVR (SEQ ID NO: 35)   -KTCTTPAQGN (SEQ ID NO: 2)   -TTPAQGN (SEQ ID NO: 37) 2. The method of claim 1, comprising less than 61 amino acids, including at least 4 amino acids of one of Polypeptide. 3. Sequence KTCTTPAQGN (SEQ ID NO: 2) or TTPAQGN (SEQ ID NO: No. 37), and the sequence FAKYWEWASVR (SEQ ID NO: 35) or the sequence thereof 2. The polypeptide of claim 1, comprising a functionally equivalent portion or variant of 4. Upon inoculation in a mammalian host, the polypeptide, in particular the sequence KTCTTPAQ GN (SEQ ID NO: 2) or a portion thereof, and the sequence TTPAQGN (SEQ ID NO: 37) Or a portion thereof, or the sequence FAKYWEWASVR (SEQ ID NO: 35) or Results in the production of an antibody that specifically binds to that portion. A repeptide or a variant thereof. 5. A polypeptide according to any one of claims 1 to 4, and a negatively charged phospholipid. Combination with. 6. A polypeptide comprising any combination of the polypeptides of claims 2-5. Composition. 7. A vaccine containing the polypeptide according to any one of claims 1 to 4 as an active ingredient. Chin composition. 8. Hepatitis B virus and / or delta hepatitis virus or any of them To vaccinate humans against infection with a mutant strain of Hepatitis B Human and / or hepatitis delta virus or any mutant thereof 8. Use as an inoculum for therapeutically vaccinating a person. A vaccine composition as described. 9. HBs specifically binding to the polypeptide according to any one of claims 1 to 4, An antibody that inhibits binding of Ag to Annexin V. 10. Hepatitis B virus and / or hepatitis delta virus or any of them 10. The method of claim 9 for use in a method of treating a human infected with one of the mutant strains. A pharmaceutical composition comprising the body or a fragment thereof as an active ingredient. 11. The polypeptide according to any one of claims 1 to 4, wherein a) HBsA. contacting a biological sample to be analyzed for the presence of the polypeptide with the polypeptide Step, b) detecting an immunological complex formed between the antibody and the polypeptide Hepatitis B and / or delta hepatitis surface treatment Antibody that is capable of competing with the proto / Annexin V interaction and is present in the biological sample Polypeptide for use in the detection method. 12. 5. At least one microplate, according to any one of claims 1 to 4, Polypeptides, appropriate buffers, binding of the polypeptides to antibodies in human serum samples Blocking and washing solutions, and antibodies in human serum samples An appropriate marker that enables measurement of a complex formed with the polypeptide. Including, hi Kit for in vitro measurement of antibodies in serum. 13. Hepatitis B virus and / or hepatitis delta virus or any of them Claims for use as a medicament for treating a human infected with one of the mutant strains Item 5. The polypeptide according to any one of Items 1 to 4. 14． About drugs that block the binding between Annexin V and polypeptides The use according to any one of claims 1 to 4, for use in a screening method. Polypeptide.